Designer Palmitoylation Motif-Based Self-Localizing Ligand for Sustained Control of Protein Localization in Living Cells and

Inducing protein translocation to the plasma membrane (PM) is an important approach for manipulating diverse signaling molecules/pathways in living cells. We previously devised a new chemogenetic system, in which a protein fused to Escherichia coli dihydrofolate reductase (eDHFR) can be rapidly translocated from the cytoplasm to the PM using a trimethoprim (TMP)-based self-localizing ligand (SL), mgcTMP. However, mgcTMP-induced protein translocation turned out to be transient and spontaneously reversed within 1 h, limiting its application. Here, we first demonstrated that the spontaneous reverse translocation was caused by cellular degradation of mgcTMP, presumably by proteases. To address this problem, we newly developed a proteolysis-resistant SL, m(D)cTMP. This m(D)cTMP now allows sustained PM localization of eDHFR-fusion proteins (over several hours to a day), and it was applicable to inducing prolonged signal activation and cell differentiation. m(D)cTMP also worked in live nematodes, making it an attractive new tool for probing and controlling living systems.